Interactions of aminoacyl-tRNA synthetases in high-molecular-weight multienzyme complexes from rat liver

The functional interaction of Arg-, Ile-, Leu-, Lys- and Met-tRNA synthetases occurring within the same rat liver multienzyme complex are investigated by examining the enzymes catalytic activities and inactivation kinetics. The Michaelis constants for amino acids, ATP and tRNAs of the dissociated aminoacyl-tRNA synthetases are not significantly different from those of the high-Mr multienzyme complex, except in a few cases where the Km values of the dissociated enzymes are higher than those of the high-Mr form. The maximal aminoacylation velocities of the individual aminoacyl-tRNA synthetases are not affected by the presence of simultaneous aminoacylation by another synthetase occurring within the same multienzyme complex. Site-specific oxidative modification by ascorbate and nonspecific thermal inactivation of synthetases in the purified rat liver 18 S synthetase complex are examined. Lys- and Arg-tRNA synthetases show remarkably parallel time-courses in both inactivation processes. Leu- and Met-tRNA synthetases also show parallel kinetics in thermal inactivation and possibly oxidative inactivation. Ile-tRNA synthetase shows little inactivation in either process. The oxidative inactivation of Lys- and Arg-tRNA synthetases can be reversed by addition of dithiothreitol. These results suggest that synthetases within the same high-Mr complex catalyze aminoacylation reactions independently; however, the stabilities of some of the synthetases in the multienzyme complex are coupled. In particular, the stability of Arg-tRNA synthetase depends appreciably on its association with fully active Lys-tRNA synthetase.     

Studies on two groups of aminoacyl-tRNA synthetases from rat liver

Aminoacyl-tRNA synthetases from rat-liver cytoplasm were fractionated into two groups, characterized by their sedimentation coefficients of about 20S and 5S, respectively. These two groups of synthetases could be isolated from postmicrosomal supernatant either by gradient centrifugation, by gel filtration or by acid treatment at pH 5.2. Both groups were required for maximal amino acid incorporation in a cell-free system.     

Glutamyl-tRNA synthetase of Escherichia coli. Isolation and primary structure of the gltX gene and homology with other aminoacyl-tRNA synthetases

The gltX gene encoding the glutamyl-tRNA synthetase of Escherichia coli and adjacent regulatory regions was isolated and sequenced. The structural gene encodes a protein of 471 amino acids whose molecular weight is 53,810. The codon usage is that of genes highly expressed in E. coli. The amino acid sequence deduced from the nucleotide sequence of the gltX gene was confirmed by mass spectrometry of large peptides derived from the glutamyl-tRNA synthetase. The observed peptides confirm 73% of the predicted sequence, including the NH2-terminal and the COOH-terminal segments. Sequence homology between the glutamyl-tRNA synthetase and other aminoacyl-tRNA synthetases of E. coli was found in four segments. Three of them are aligned in the same order in all the synthetases where they are present, but the intersegment spacings are not constant; these ordered segments may come from a progenitor to which other domains were added. Starting from the NH2-end, the first two segments are part of a longer region of homology with the glutaminyl-tRNA synthetase, without need for gaps; its size, about 100 amino acids, is typical of a single folding domain. In the first segment, containing sequences homologous to the HIGH consensus, the homology is consistent with the following evolutionary linkage: gltX----glnS----metS----ileS and tyrS.     

Macromolecular complexes of aminoacyl-tRNA synthetases from eukaryotes. 1. Extensive purification and characterization of the high-molecular-weight complex(es) of seven aminoacyl-tRNA synthetases from sheep liver.

Starting from homogenates of sheep liver, extensive co-purification of seven aminoacyl-tRNA synthetases to high specific activities was achieved by a three-step procedure involving fractional precipitation by poly(ethylene glycol) 6000, gel filtration on 6% agarose and chromatography on Sepharose-bound tRNA. The purified material is composed of nine major protein components as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and has an apparent molecular weight of about 10(6) estimated by gel filtration on 6% agarose. It contains aminoacyl-tRNA synthetase activities specific for methionine, lysine, arginine, leucine, isoleucine, glutamine and glutamic acid. The rigorous co-elution of these seven enzymes at each chromatographic step suggests, but does not conclusively prove, that they are physically associated within the same complex. The enzyme composition of the high-molecular-weight complex purified from sheep liver is identical to that of the complex previously isolated from human placenta by Denney in 1977 (Arch. Biochem. Biophys. 183, 156--167).     

Characterization of a proteolipid complex of aminoacyl-tRNA synthetases and transfer RNA from rat liver.

A high molecular weight complex containing aminoacyl-tRNA synthetases, peptidyl acetyltransferase, lipids and tRNA has been isolated from the 250,000 x g postmitochondrial supernatant from rat liver cells. Aminoacyl-tRNA synthetase activity directed towards arginine, aspartate, glutamine, glutamate, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, and tyrosine is present. An endogenous pool of aminoacyladenylates is indicated by an ATP-32PPi exchange catalyzed by the native complex, which shows a dramatic increase after addition of ATP. Lysine is the only amino acid which greatly increases the exchange rate catalyzed by the native complex in vitro, whereas components of the denatured complex activate all the 13 amino acids in the presence of ATP. Six of the eight lipid fractions were glycolipids; cholesterol and cholesterol esters were absent. The extracted RNA has many characteristics of tRNA. These findings provide evidence for the organization of aminoacyl-tRNA synthetases in a complex with peptidyl acetyltransferase that also contains lipids and tRNA and that can be readily isolated from the cytosol of rat liver cells.     

Occurrence of 5SrRNA in high molecular weight complexes of aminoacyl-tRNA synthetases in a rat liver supernatant.

The 5SrRNA in the rat liver postmicrosomal supernatant was investigated. Acrylamide gel electrophoresis and Northern blot analysis showed that most of the 5SrRNA was present in the fractions obtained on high molecular weight regions separated by Sephadex G-200 column chromatography of the supernatant, which contained the bulk of the methionyl-tRNA synthetase (Fraction I) and tyrosyl-tRNA synthetase (Fraction II). A high molecular weight complex containing nine aminoacyl-tRNA synthetases [Mirande, M., LeCorre, D., & Waller, J.-P. (1985) Eur. J. Biochem. 147, 281-289] was purified by fractional precipitation with polyethylene glycol 6000, gel filtration on Bio-Gel A-1.5m, and finally tRNA-Sepharose column chromatography, which gave two fractions. Fraction B showed the activities of nine aminoacyl-tRNA synthetases and gave protein bands corresponding to eight previously identified enzymes on SDS-PAGE. Fraction A, eluted with a lower KCl concentration than Fraction B, showed lower activities than fraction B of eight of the aminoacyl-tRNA synthetases, the exception being prolyl-tRNA synthetase. The staining patterns with ethidium bromide of the RNAs after PAGE showed 5SrRNA bands for Fraction A but not for Fraction B. However, Northern blot analysis indicated that 5SrRNA was present in both Fractions A and B. The staining pattern after SDS-PAGE of Fraction A with Coomassie Brilliant Blue showed several protein bands in addition to those observed for Fraction B, one of which, with a staining intensity comparable with those of other bands, was located at the same position as ribosomal protein L5, which is the protein moiety of the 5SrRNA-L5 protein complex of ribosomal 60S subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the evolution of structure in aminoacyl-tRNA synthetases.

The aminoacyl-tRNA synthetases are one of the major protein components in the translation machinery. These essential proteins are found in all forms of life and are responsible for charging their cognate tRNAs with the correct amino acid. The evolution of the tRNA synthetases is of fundamental importance with respect to the nature of the biological cell and the transition from an RNA world to the modern world dominated by protein-enzymes. We present a structure-based phylogeny of the aminoacyl-tRNA synthetases. By using structural alignments of all of the aminoacyl-tRNA synthetases of known structure in combination with a new measure of structural homology, we have reconstructed the evolutionary history of these proteins. In order to derive unbiased statistics from the structural alignments, we introduce a multidimensional QR factorization which produces a nonredundant set of structures. Since protein structure is more highly conserved than protein sequence, this study has allowed us to glimpse the evolution of protein structure that predates the root of the universal phylogenetic tree. The extensive sequence-based phylogenetic analysis of the tRNA synthetases (Woese et al., Microbiol. Mol. Biol. Rev. 64:202-236, 2000) has further enabled us to reconstruct the complete evolutionary profile of these proteins and to make connections between major evolutionary events and the resulting changes in protein shape. We also discuss the effect of functional specificity on protein shape over the complex evolutionary course of the tRNA synthetases.     

Affinity chromatography of rat liver aminoacyl-tRNA synthetase complex

The affinity column lysyldiaminohexyl-Sepharose 4B has been synthesized for the purification of aminoacyl-tRNA synthetase complexes. Lysyl-tRNA synthetase (EC 6.1.1.6) bound specifically to the Sepharose-bound lysine. The purified lysyl-tRNA synthetase was associated with arginyl-tRNA synthetase (EC 6.1.1.16) and sedimented at 18S and 12S. A 24S lysyl-tRNA synthetase bound specifically to the affinity column and also found associated with arginyl-tRNA synthetase. The results favor the model of a heterotypic multienzyme complex of mammalian aminoacyl-tRNA synthetases.     

A role for lipids in the functional and structural properties of the rat liver aminoacyl-tRNA synthetase complex

The high molecular weight aminoacyl-tRNA synthetase complexes found in extracts of many eukaryotic cells often contain lipids and other non-protein components. Since hydrophobic interactions play an important role in maintaining synthetases in the complex, it has been suggested that the lipids present may also participate in its functional and structural integrity. In order to learn more about the role of lipids in the complex, we have compared the properties of the normal complex to one which has been delipidated by treatment with Triton X-114. Delipidation does not affect the size or activity of the aminoacyl-tRNA synthetase complex, but a variety of functional and structural properties of individual synthetases in the complex are altered dramatically. These include sensitivity to salts plus detergents, temperature inactivation, hydrophobicity, and sensitivity to protease digestion. In the latter case, removal of lipids also affects the low molecular weight products released by protease digestion. Purification of the synthetase complex by various chromatographic procedures can remove the lipids and lead to a structure that behaves like the delipidated complex prepared by detergent treatment. The significance of these findings for the intracellular location of aminoacyl-tRNA synthetases and for the study of purified complexes are discussed.     

Molecular weights of mitochondrial and cytoplasmic aminoacyl-tRNA synthetases of beef liver and their complexes

In eukaryotes, multienzyme complexes containing five to nine aminoacyl-tRNA synthetase activities have frequently been reported. In this study, we report the existence, in bovine liver cytoplasm, of a multienzyme complex containing at least 16 activities which can be disrupted by homogenization to give rise to smaller complexes and noncomplexed synthetases. Determination of the size and component activity of these complexes and of the molecular weights of all 20 free synthetases suggests that the smaller complexes and free activities normally identified arise from the larger complex by well-defined stages during homogenization. We also show that similar, though not identical, complexes are found in bovine liver mitochondria and give the molecular weights of 16 mitochondrial synthetases.     

Chromatofocusing of aminoacyl-tRNA synthetases, extracted from NMRI mouse liver

Chromatofocusing of 17 aminoacyl-tRNA synthetases extracted from NMRI mouse liver is described and the apparent isoelectric points of these enzymes are presented. Each of 15 aminoacyl-tRNA synthetases was present in two peaks. Isoleucyl-tRNA synthetase showed only one peak and arginyl-tRNA synthetase was present in three peaks. Phosphorylation/dephosphorylation experiments with arginyl-tRNA synthetase indicate that the peaks represent phosphorylated and unphosphorylated synthetase protein. One example of detection of increased protein phosphorylation during a biological experiment is presented.     

The plant aminoacyl-tRNA synthetases. Effect of sodium chloride on tRNA aminoacylation and aminoacyl-tRNA decomposition catalysed by aminoacyl-tRNA synthetases from yellow lupin seeds.

The initial velocity and the extent of aminoacylation are affected by sodium chloride in the lupin aminoacylation systems involving serine, isoleucine, lysine, leucine, phenylalanine and valine. Pyrophosphorolysis and enzymatic hydrolysis of [14C]Val-tRNA catalysed by lupin valyl-tRNA synthetase are inhibited by sodium chloride nearly to the same extent. Evidence is presented that when a limiting amount of synthetase is used, the equilibrium of the aminoacylation reaction in the lupin valine system is determined only by the rate of aminoacylation and non-enzymatic deacylation of aminoacyl-tRNA, the former but not the latter reaction being dependent on concentration of the enzyme and monovalent salt.     

Renaturation of rabbit liver aminoacyl-tRNA synthetases by 80S ribosomes.

Protein biosynthesis machinery is thought to be mostly compartmentalised within the mammalian cell, involving direct interactions between different components of the translation apparatus. The present research concerns the functional meaning of the interaction between the rabbit liver aminoacyl-tRNA synthetases and 80S ribosomes. We have shown that rabbit liver 80S ribosomes are able to enhance the activity of leucyl-tRNA synthetase, which is a component of high-molecular weight aminoacyl-tRNA synthetase complex, and phenylalanyl-tRNA synthetase not associated within this complex. The ribosomes increase the initial rate of both the total reaction of tRNA aminoacylation and the first step of this reaction, the formation of leucyladenylate. Moreover, a positive cooperativity of the tRNA interaction with two binding sites of leucyl-tRNA synthetase is also increased in the presence of highly purified 80S ribosomes. The effect of 80S ribosomes on partly denatured leucyl-tRNA synthetase and phenylalanyl-tRNA synthetase and the protection by 80S ribosomes of both enzymes against inactivation indicate a refolding and stabilising capacity of the ribosomes. It is concluded that the interaction of aminoacyl-tRNA synthetases and 80S ribosomes is important for the maintenance of an active conformation of the enzymes.